Abstract

Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH), defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity, and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO signaling. We identified 2 pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.

Figure 5

(A) Reaction sequence of oxyHb with NO under conditions of excess NO. In a 3-step reaction, oxyHb can scavenge up to 3 molecules of NO. (B) NO-reaction kinetics of Hb (left), Hb-haptoglobin (middle), or polymerized Hb (right). Shown are absorbance changes at 405 nm after rapid mixing of NO and oxyHb in a stopped-flow spectrophotometer. Estimates of the reaction rates (k1 and k2) were calculated by approximating the data with an equation defined by the sum of 3 exponential functions. (C) Tension traces (mean ± SD) of porcine basilar arteries immersed in buffer containing 10 μM Hb (left; red, n = 16), Hb-haptoglobin (middle; green, n = 16), or polymerized Hb (right, brown, n = 8). A bolus of MAHMA-NONOate was added (arrowhead) to induce NO-mediated dilation. (D) Coronal vibratome sections (2 mm anterior to the bregma) of mouse brains 2 hours after subarachnoid injection of TCO-labeled Hb (left), Hb-haptoglobin (middle) and polymerized Hb (right). The false-colored images represent the signal intensity of the injected compound after postmortem coupling to a fluorophore (tetrazine-5-TAMRA). Intraparenchymal delocalization is only observed in the mouse injected with Hb and not after injection of Hb-haptoglobin and polymerized Hb (images are representative for n = 3 per group). The signal in the area of the puncture channel (dashed boxes) serves as a positive control for the injection and labeling procedure. (E) SEC elution profiles of pure Hb (left; red), Hb-haptoglobin (middle; green), and polymerized Hb (right; brown) measured at 414 nm illustrate the different molecular size of the injected compounds.